CK는 컬럼비아 대학 대학원 프로그램에 의해 지원됩니다. UB는 미국의 백혈병과 림프종 사회, 백혈병 연구 재단의 새로운 탐정 수상의 수신자의 연구원이며, 컬럼비아 대학 뉴 학부 시동 자금에 의해 지원됩니다.

1. 지라 B - 림프구 분리 및 자극 <ol><li> AID - 결함 쥐들이 12 세 2~3개월 사이 spleens를 수확. 비장의 분리는 무균 상태에서 수행되고 장기가 일시적으로 15% 태아 소 혈청 (FBS)이있는 차가운 인산 버퍼 호수 (PBS)에 보관됩니다. </li><li> 비장은 다음 멸균 조직 문화 후드로 전송하고 2.5 % FBS와 보충 PBS 5 ML에 균질입니다. 15 ML 팔콘 튜브에 homogenate를 전송합니다. </li><li> 4 10 분 동안 1000rpm에서 균질 지라 조직 샘플을 원심 분리기 ° C를 펠릿 세포 수 있습니다. </li><li> 원심 분리 후 뜨는을 가만히 따르다. 적혈구에 의해 오염되지 않도록 10 ML 붉은 혈액 세포에서 세포 펠릿 (RBC) 7 분 상온에서 용해 버퍼를 Resuspend. </li><li> 10 분 가만히 따르다 뜨는 1,000 RPM에서 샘플을 원심 분리기 10 ML PBS 2.5 % FBS를 포함하는에있는 세포를 resuspend. </li><li> 100 μL 나누어지는를 제거하고 죽은 세포를 제외 trypan 파랑을 사용하여 표준 hemacytometer를 사용하여 1:1000 희석에서 세포를 계산합니다. 약 20-30000000 세포가 비장마다 얻을 수 있습니다. </li><li> 10 분 1000rpm에서 샘플을 원심 분리기 및 뜨는을 가만히 따르다. </li><li> 0.5 % 90 μL 당 10 7 세포의 농도에서 BSA / PBS 포함 2mM EDTA (에틸렌 다이아 민 테트라 초산)에서 펠렛을 Resuspend. </li><li> 특히 비 - B 세포를 바인딩하는 세포 방지 CD43 항체 코팅 자기 구슬을 추가합니다. 90 μL 당 10 μL 비즈 (10 7 세포)를 사용합니다. 잘 믹스 4에서 품어 ° C를 20 분. </li><li> 세포로 분류되었습니다되면, FBS / PBS는 튜브 맨 위로 2.5 %를 추가하여 그들을 씻으십시오. 10 분 1,000 RPM에서 샘플을 원심 분리기. </li><li> 뜨는을 가만히 따르다. ML 당 10x10 7 세포의 농도에서 0.1 % BSA / PBS에 세포를 Resuspend. </li><li> 자기 분리기에 자기 정렬 칼럼을 마운트합니다. 흐름 -을 통해 수집하고 BSA / PBS 총리와 씻어 3mL 0.5 %로 컬럼을 읽어 직접 열을 아래 원뿔 튜브를 놓습니다. </li><li> 세척 유체가 통과 거듭되면, 새 컬렉션 튜브로 교체하십시오. 샘플 미만 1 ML 경우, 0.5 % BSA / PBS / EDTA (에틸렌 다이아 민 테트라 초산) 1 ML 샘플 볼륨을 조절하고 열에있는 샘플을로드합니다. 칼럼 당 세포의 1 ML 최대를로드하고 필요한 경우 여러 개의 컬럼을 사용합니다. </li><li> 세포가 수동적으로 열의 컬럼을 통해 흐름과 원뿔 관에 언바운드 세포를 수집하도록 허용합니다. 0.5 %는 BSA / PBS -을 통해 플로우를 수집하기 위해 지속하면서 3 ML과 열 4 번 씻으십시오. </li><li> 모두 CD43 음성 쉬고있는 B 세포가 풍부하게 될있는 흐름을 통해 수집합니다. 열을 삭제. 10 분 1,000 RPM의 흐름을 통해 원심 분리기. </li><li> 뜨는 드레인 10 ML 매체 (15 % FCS / + 글루타민, 펜 / Strep, 비 필수 아미노산, 50 μm의 Β - ME)를 추가 </li><li> ML 당 10 6 세포에있는 세포와 문화를 계산합니다. </li><li> B 세포 성장과 확산을 촉진하기 위해, 각각 최종 농도 20 μg / ML 및 1 μg / ML하는 등 재조합 IL - 4 및 안티 CD40 항체와 매체를 보완하고, 문화 플라스크에 세포를 전송합니다. </li><li> 하룻밤 문화 셀을. 세포를 카운트하고 적절한 IL - 4 및 CD40를 추가, ML 당 10 6 세포로 희석. </li><li> 바이러스 감염 전에 72 시간 문화 전지. </li></ol> 2. 제작자 세포 Transfection 양이온 lipids 또는 인산 칼슘을 사용하여 transfection을 위해 설립 프로토콜을 사용합니다. 접시 당 100 μg 플라스미드 DNA를 사용하여 DNA 구조 당 Transfect의 10cm 3 판. 우리는 일상적으로 칼슘 인산염 transfection 방법 13, 14을 사용하고 chloroquine과 함께 보충 때 높은 바이러스 titer를 적어 둡니다. <ol><li> 생산자 세포가 transfected 수로 60%에게 70 % 합류 피닉스 세포를 사용합니다. 이 코플루엔스 셀 카운트에서 10cm 접시 당 5,000,000 전지해야합니다. </li><li> transfection하기 전에 한 시간, 피닉스 세포 성장을위한 새로운 완전한 미디어로 미디어를 교체하십시오. </li><li> 멸균 eppendorpf 관에서 포장 구조 DNA, 0.5M CaCl 2 250 μL 100 μg을 결합하고, 멸균 물 500 μL에 최종 음량을 조절 </li><li> 1 ML 피펫을 사용하여 적극적으로 15mL 폴리 프로필렌 튜브 500 μL 배 HNP이 솔루션을 믹스 </li><li> 이 혼합물은 8-10분에 대한 실온에서 휴식을 허용합니다. 이 기간 동안 침전물이 형성됩니다. </li><li> transfection 혼합 균일 내내 혼합물을 배포하는 매체를 소용돌이 치는 동안 세포 드롭 현명한을 추가합니다. </li><li> 37 세포를 품어 ° C를 12 시간. </li><li> 12시간 포스트 transfection은 8ml 전체 B 세포 성장 매체와 매체를 교체하고 37 ° C 배양기에 세포를 반환합니다. </li></ol> 3. 자극 지라 B 세포 감염 <ol><li> 포스트 transfe 48hr 피닉스 세포의 표면에 뜨는 제거ction 및 세포 파편을 제거하는 0.2 미크론 필터를 통해 뜨는을 전달합니다. </li><li> 3 ML 믹스 바이러스를 포함하는 미리 활성화 B 세포 (1 단계에서와 같이) 3 ML로 뜨는 있습니다. 세포에 바이러스 흡착을 향상시키기 위해서는, ML 당 16 μg의 최종 농도에 polybrene를 추가합니다. </li><li> 30 90 분간 2500 rpm으로 원심 분리하여 세포를 감염 ° C. </li><li> 37 이상 48-72 시간에 대한 품어 스핀, ° C는 세포 성장과 확산 수 있도록 즉시. </li></ol> 4. 유동세포계측법 분석 <ol><li> 흐름 cytometer를 사용하여, B220 및 표면 IgG1의 표면 표현에 대한 세포를 분석할 수 있습니다. 세포의 표면에서 IgG1의 존재는 그 클래스 스위치 재조합이 AID 유전자의 retroviral complementation을 통해 성공적인 구조의 결과로 발생했습니다 나타냅니다. </li></ol> 5. 대표 결과 우리는 성공적으로 (AID - / -) AID - 결함에서 단백질 요소 활성화 유도 Cytidine의 Deaminase (AID)의 결핍을 구출해야 retroviral 전달을 사용하는 B - lymphocytes. 간단히, 우리는 pMX - 하녀 - GFP 15-18 피닉스 세포 transfecting하여 마우스 AID (하녀) 단백질을 표현하는 재조합 retroviruses를 생성. 수확 바이러스는 전 생체내 클래스 스위치 재조합 (CSR) 자극 조건 19 AID - 부족한 생쥐에서 얻은 AID - 결함 B 세포에 감염되었습니다. 감염된 B 세포는 다음 유동세포계측법 분석을 사용하는 문화에 72hrs 이후 CSR에 대한 IgG1을 분석했다. 그림 2는 CSR는 원조 표현 구조의 결과로 발생했음을 나타냅니다 AID가 아니라 비어있는 구조와 B 세포의 표면에 IgG1의 감지를 보여줍니다. <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="그림 1" /> 그림 1 : 생산 세포 내에서 retroviral 포장 제도 : 이전 15 설명된대로 Ψ로 표시 관심있는 유전자가,,, pMX - IRES - GFP에 subcloned되었습니다. 플라스미드는 인산 칼슘 방법 13, 14을 사용하여, 개그 - POL 및 봉투 단백질 (피닉스, Orbigen)를 생산 할 수 ecotropic 바이러스성 포장 세포주에 transfected되었습니다. 마흔 여덟 시간 transfection 후 뜨는 포함하는 바이러스성 입자는 생쥐에서 얻은 대상 기본 B 세포에 감염 수확했다. <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="그림 2" /> 그림 2 : AID 결함 B 세포는 안티 CD40로 자극하고 IL - 4 pMX - 메이드 - GFP 또는 혼자 GFP를 표현하는 레트로 바이러스를 포함하는 레트로 바이러스에 감염되었습니다. IgG1에 CSR의 수준은 외과의 분석에 의해 평가되었다.

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관심 없음 충돌 선언하지 않습니다.

lymphopoesis의 발달 이벤트의 많은가 transcriptional 규칙 1, 2에 의해 제어되기 때문에 그림 1에 여기에서 설명한과 같이 묘사 같은 지라 B 세포의 Retroviral 도입은 B - lymphocytes의 연구에 유용 유전자 접근 방식이다. CD40L를 통해 트리거링 B 세포 성숙의 나중 단계에서 B 세포 성장의 유도, 세포주기에 진입하고, 증식 11, 20 필수입니다. 그림 2에 표시된, B 세포는 확?...

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<td>FCS</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
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<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>22400</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>20012</td>
<td>&nbsp;</td>
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<td>Red Blood Cell (RBC) lysis buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>CD43 beads</td>
<td>&nbsp;</td>
<td>Miltenyi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B Cell Complete Media</td>
<td>&nbsp;</td>
<td>Various components</td>
<td>Various Components</td>
<td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&mu;M &beta;-Mercaptoethanol</td>
</tr>
<tr>
<td>IL-4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD40</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>553787</td>
<td>&nbsp;</td>
<tr>
<td>polybrene</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>107689 &#x2028;</td>
<td>&nbsp;</td>
<tr>
<td>Chloroquine diphosphate salt</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>C6628</td>
<td>Used at 100mM</td>
</tr>
<tr>
<td>Phoenix Eco cells (Murine)</td>
<td>&nbsp;</td>
<td>Orbigen</td>
<td>RVC-10002</td>
<td>&nbsp;</td>
<tr>
<td>PE-Cy5-&alpha;-mouse-CD45R (B220)</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>15-0452-81</td>
<td>&nbsp;</td>
<tr>
<td>PE-&alpha;-mouse-IgG1</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>A85-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

recombinant retroviral production and infection of b cells

진핵 세포에있는 유전자의 형질 표현 관심의 유전자가 결과의 표현형 분석을 용이하게하기 위해 heterologous 표현 시스템의 제어하에 표현되는 강력한 반대 유전자 접근 방식이다. 이 접근 방법은 일반적으로 유기체에서 발견되지 않은 유전자를 표현하는 유전자 제품의 돌연변이 형태를 표현하기 위해, 또는 유전자 제품의 지배 - 부정적인 양식을 통해 - 표현하는 데 사용될 수 있습니다. 그것은 transcriptional 규정이 2-4 검토 B 세포 1의 개발과 차별화의 주요 제어 메커니즘입니다 hematopoetic 시스템의 연구에서 특히 유용합니다. 마우스 유전학은 인간의 유전자와 질병의 연구를위한 강력한 도구입니다. 마우스와 인간 게놈의 비교 분석은 게놈 5 90 % 이상의 synteny의 절약을 보여준다. 또한, 많은 마우스 모델에 사용되는 기술의 예를 들어, 유전자 중단 및 allelic 교체 6 인간의 유전자 연구에 적용 . 그러나, 유전자 변형 마우스의 생성은 재정 및 기술 자연 모두 자원의 큰 거래를 필요로합니다. 몇몇 프로젝트는 마우스 변종을 노크의 라이브러리 (KOMP, EUCOMM, NorCOMM) 또는 대규모 노력과 협업 7 필요한 돌연변이 유발 유도 변종 (RIKEN)를 컴파일하기 시작했다. 따라서, 먼저 마우스 모델에 진행하기 전에 기본 세포의 세포 배양 모델에 원하는 유전자의 표현형 연구를하는 것이 바람직하다. Retroviral DNA는 안정의 결과, 선호 내에서 또는 전사 단위 또는 CPG 섬으로 근처 호스트 DNA에 통합하여 transcriptional 입을 8 9을 회피하면서 관심의 포장 유전자의 표현을 물려 전할 수있는. 유전자는 다음 전사 및 단백질 생산의 높은 효율성의 결과, 높은 효율 retroviral 발기인의 통제하에 베꼈는데 있습니다. 따라서, retroviral 표현은 transfect하기 어려운 세포와 함께 사용 유사 분열 동안 활성 상태에있는 세포를 제공할 수 있습니다. 바이러스의 구조 유전자가 패키징 세포주에 포함된 있기 때문에, 관심있는 유전자를 복제하는 데 사용되는 표현 벡터는 모두 바이러스 revertants의 가능성을 제거하고 바이러스 supernatants 작업의 안전을 증가하는, 바이러스에 대한 구조 유전자를 포함하지 더 전염성 virions는 10을 생산하지 않습니다로. 여기 재조합 retroviral 생산과 지라 B 세포의 후속 감염에 대한 프로토콜을 제시한다. 분리 후, 교양 지라 세포 목 파생 lymphokines 및 B 세포의 증식과 분화 11 파열을 유도 방지 CD40과 자극하고 있습니다. 이 프로토콜은 B 세포가 plasmacytic 분화를 유도하는 것을 원칙으로하지만 이전 antigenic의 자극에 초기 hematopoetic 이벤트 다음과 비장으로부터 격리되면서, 늦은 B 세포 개발과 차별에서 발생 사건의 연구에 이상적입니다.

Watch this Scientific Journal Video about Recombinant Retroviral Production and Infection of B Cells at JoVE.com

Recombinant Retroviral Production and Infection of B Cells

에서 유전자의 구조와 기능 분석의 효율적인 시스템<em&gt; 예 생체내</em지라 B - lymphocytes의&gt; 문화 설명되어 있습니다. 이 메서드는 도우미 무료 ecotrophic 포장 세포주에서 재조합 retroviral 생산을 활용합니다. 안정적인 수준의 스위치 재조합을받은 B 세포 표면 항체의 생성으로 이어지는 이루어집니다 주 lymphocytes 이내에 관심이 유전자의 표현을 물려 전할 수있는.

B 세포의 재조합 생산 및 Retroviral 감염

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

recombinant-retroviral-production-and-infection-of-b-cells

Research

JoVE Journal

Immunology and Infection

14.0K 조회수.  Columbia University College of Physicians and Surgeons. 에서 유전자의 구조와 기능 분석의 효율적인 시스템 예 생체내 문화 설명되어 있습니다. 이 메서드는 도우미 무료 ecotrophic 포장 세포주에서 재조합 retroviral 생산을 활용합니다. 안정적인 수준의 스위치 재조합을받은 B 세포 표면 항체의 생성으로 이어지는 이루어집니다 주 lymphocytes 이내에 관심이 유전자의 표현을 물려 전할 수있...

비디오: Recombinant Retroviral Production and Infection of B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

연구

면역학 및 감염병학

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

재조합 Adeno - 관련 바이러스성 벡터의 생산 및 Titering

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

아데노 바이러스 파생 시스템의 도움으로 효율적인 재조합 파르 보 바이러스 생산

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

진정한 검출 마우스 B 리니지 세포를 IgE 항체가 발현

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

정렬 된 불멸화 된 B 세포로부터 재조합 인간 IgG 단일 클론 항체의 생성

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

재조합 형 단백질의 생체 외에서 사이트별 Glycosylation에 대 한 대상 시스테인 Thiols

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

기본 Hepatocytes 및 B 형 간염 바이러스 감염에 대 한 Kupffer 세포의 "간-온-어-칩" 문화

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

뮤린 B-1a 세포에 CXCR4의 레트로 바이러스 과발현 및 골수와 IgM 생산에 표적 B-1a 세포 이동을 위한 채택 전송

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...